Orientia tsutsugamushi in chiggers and small mammals in Laos

Scrub typhus is a leading cause of febrile illness in Laos and accounts for a high burden of disease. There have been no previous studies on the causative agent, Orientia tsutsugamushi, in vector mites (“chiggers”) or their small mammal hosts in Laos. Small mammals and free-living chiggers were trapped in districts of Vientiane Province and Capital. Tissues were tested for O. tsutsugamushi by PCR and serum for IgG to O. tsutsugamushi by immunofluorescence assays (IFA). Chiggers removed from small mammals and collected in their free-living stage using black plates were identified and tested for O. tsutsugamushi by PCR. Over an 18-month period 131 small mammals of 14 species were collected in 5 districts. Seventy-eight of 131 small mammals were infested with chiggers, but all tissues were O. tsutsugamushi PCR negative. Eighteen species of chigger were identified and 1,609 were tested by PCR. A single pool of chiggers tested O. tsutsugamushi positive. Serum from 52 small mammals were tested by IFA, with 16 testing positive. These are the first molecular and serological data on O. tsutsugamushi in chiggers and small mammals in Laos. Further studies are needed to better understand the key vector species and ecology of scrub typhus in areas with high disease incidence in Laos.


Introduction
Scrub typhus is a potentially fatal neglected tropical disease caused by the obligate intracellular bacterium Orientia tsutsugamushi. The pathogen is transmitted by the bite of larval stage trombiculid mites (chiggers) that are predominantly hosted by small mammals [1]. The disease is endemic to the "tsutsugamushi triangle" region of the Asia-Pacific, and threatens over a billion people, leading to severe febrile illness, with complications including pneumonitis, meningoencephalitis, myocarditis and renal failure [2]. The median untreated mortality is ˜6% [3]. Rural workers and military personnel are typically at highest risk, but cases are increasing among city dwellers visiting the countryside for recreation [4].
The Lao PDR (Laos) is a lower-middle income, land-linked country in Southeast Asia with a population of close to 7 million people. Two thirds of the population live in rural areas and half of these are subsistence farmers [5]. Scrub typhus was first reported in Laos in 1939 [6], but not subsequently reported until 2006 [7]. The disease is a leading cause of febrile illness in Laos, accounting for 14.8% of admissions with community-acquired septicaemia to hospital in the capital, Vientiane [7]. Seroprevalence in the capital was 20% and is likely to be higher in more rural populations [8]. Spatiotemporal analysis of scrub typhus showed a negative association with the more urbanized parts of the capital city and a strong seasonal pattern, with cases peaking in the wettest months of July-September [9].
For the first time in Laos, we investigate for the presence of O. tsutsugamushi in chiggers and small mammal hosts at sites of high-disease transmission across Vientiane Province. We describe the species composition, dynamics and rates of O. tsutsugamushi infection in chiggers and small mammals and relate these data to studies performed in the region.

Study sites
Four districts of Vientiane Province and one district of Vientiane Capital, Lao PDR were selected for investigation of small mammals and chiggers. These were selected due to a high incidence of scrub typhus [9] and recently identified scrub typhus cases with evidence of exposure in peri-village habitats. Small mammal trapping took place at several locations in each district, selected and performed by local hunters experienced in trapping small mammals.

Small mammal and chigger collection
Small mammals were trapped alive using locally made wire-mesh traps baited with a range of food items by local hunters. Small mammal trapping took place in the hot, dry season of May 2015, cool dry season of February 2016 and wet season of October 2016.
Free-living chiggers were collected using the black plate method [10,11]. Black acrylic plates (10 x 30 cm) were placed on the ground for 10 minutes and examined using a 30-40x hand lens for chiggers every 2 minutes. Chiggers were collected using a pipette and transferred into a sterile tube containing 70% ethanol. The location of all collected field samples were recorded using a Garmin Oregon (Garmin International, Olathe, Kansas) GPS device (accurate to ~3 metres).

Small mammal and chigger identification and processing
Traps containing small mammals were transferred to a mobile laboratory and placed in an airtight box with the addition of cotton wool soaked with 10 ml of the inhalational anaesthetic isoflurane. Animals were only removed from the trap once dead. All animal handling and euthanasia procedures followed international guidelines [12,13]. All field and mobile laboratory protocols and procedures followed the "Protocols for field and laboratory rodent studies" 2011 guide [14]. This study was approved by the Oxford Tropical Research Ethics Committee (OxTREC 48- 15 & 52-14) and by the National Health Ethics Committee, Laos (039/2016/NEHCR).
The methods used follow those described in Elliott et al [15]. In brief, small mammals were weighed in grams and the head and body, tail, hind foot, ear and skull measured in millimetres and identified to species level wherever possible following the keys in Chaval, 2011 [16]. Prior to harvesting tissues, scissors and forceps were thoroughly washed consecutively in Dettol (chloroxylenol), sterile water and 70% ethanol to prevent crosscontamination. Small pieces (<50 mg) of lung, liver and spleen were collected into prelabelled cryotubes and stored on dry ice in the field and subsequently at -80°C in the laboratory.
Both ears were detached as close to the skull as possible and stored in 70% ethanol at +4°C. The rest of the animal was examined for chiggers, notably the posterior legs, ventral midline and anogenital region. In the laboratory the ears or skin were examined under a Brunel IMXZ stereo microscope (Brunel Microscopes Ltd., Chippenham, U.K.) at 15x to 100x magnification. Approximately 15% of chiggers on each animal were identified to species. These were selected based on different appearances under lower-power microscopy in an attempt to capture the diversity of species present on the host. Chiggers were placed between two cover slips on a glass slide and morphological identification to species was performed using a Nikon Eclipse 80i compound microscope at 400x or 600x. Images were viewed and saved using the Nikon NIS Elements D 4.13.05 software package. Both autofluorescence and bright-field microscopy techniques were used [17]. A scale bar was applied to each image. All morphometric measurements and image manipulation was performed using ImageJ (https://imagej.net/ImageJ). The genus was identified with reference to Nadchatram & Dohany 1974 [18] and Vercammen-Grandjean 1968 keys to Southeast Asian chigger Elliott  genera [19]. A wide range of taxonomic identification keys were employed to identify to species level [20][21][22][23].

Small mammal and chigger DNA extraction and PCR
DNA was extracted from individual chiggers, pools of chiggers (~20-50 unidentified individuals of possible multispecies from the same host animal) and rodent tissues using the Qiagen Blood and Tissue Kit (Qiagen, USA). Chiggers were rinsed with distilled water and individuals cut through the mid-gut using a sterile 30G needle under a dissecting microscope. Pools were crushed using a sterile polypropylene motorized pestle (motorized pellet pestle Z35991, Sigma Aldrich, St Louis, MO). Rodent tissues were cut into a small piece (≤10mg of spleen or ≤25mg of liver or lung). Samples were incubated with proteinase K at 56°C for 3 hours. The rest of the steps followed the manufacturer's protocol. Chigger samples were eluted in 45μl and rodent samples in 100μl of buffer AE (Qiagen, Hilden, Germany). Samples were stored at -20°C before PCR. Real-time PCR targeting the 47kDa O. tsutsugamushi outer-membrane protein was performed on all samples as previously described [24], except for chigger samples where 5 μl of DNA template was added. PCR was run on a Bio Rad CFX96 (Bio Rad, USA).

Small mammal serology
The Scrub typhus "gold standard" immunofluorescent assay (IFA) was used to determine the endpoint IgG antibody titer in the rodent samples with the full methodology described elsewhere [25]. Briefly, 40-well slides, produced in-house at the Mahidol-Oxford Tropical Medicine Research Unit (MORU), Bangkok, Thailand, were coated and fixed with typespecific O. tsutsugamushi antigen strains (Karp, Kato, Gilliam, and TA716). Two-fold serial dilutions of rodent serum from 1:16 to 1:4096 were prepared in 2% skim milk in phosphate buffered saline (PBS) diluent. 2 μl of diluted serum was dropped onto each well and incubated at 37°C for 30 minutes. The slide was then washed four times with PBS, dried and 2 μl of diluted FITC anti-mouse conjugate added to each well. The incubation and washing steps were then repeated and fluorescence mounting medium added to the slide. Two experienced technicians examined the slides to determine the end-point titre as the highest titer displaying specific fluorescence. Samples with a titer ≥1:16 were considered positive for the presence of IgG antibodies [26,27].

Results
Four districts of Vientiane Province were visited. Small mammals were trapped at 10 sites in Phonehong (18 1). Sites in eastern Thoulakhom were in upland terrain between 600 and 850 metres above mean sea level (msl). The habitat consists of upland rice fields, fallow areas, bamboo and degraded secondary forest. All other sites were in lowland areas between 170 and 250m above msl, formed of lowland rice paddies, fallow areas and fruit plantations.
A total of 131 small mammals were collected during 3 visits over 18 months. These comprised 14 species of Rodentia and 1 species of Scandentia (Tupaia glis) (    [28,29] and in relation to the recently described Laotian rock-rat, Laonastes aenigmanus [30]. Here we report the first identification of O. tsutsugamushi in chiggers in Laos and a description of chigger species collected at sites across Vientiane Province, close to the capital city. positive. This gives an unexpectedly low positivity rate of 1.6% of chigger pools. In previous studies, infection rates using molecular methods have ranged from 0.6% to 5% in individual chiggers, 9.6 to 56% in chigger pools and 10% of small mammals [1]. These low rates may be accounted for by several factors. A relatively low number of small mammals were trapped in this study. Rodent capture rates are known to be lower in Laos compared to neighbouring Thailand and Cambodia [31]. This observation remains difficult to explain, but may be due to high hunting intensity by local communities and that capture rates are influenced by seasonal climatic conditions and climatic variability. Two of the three collection periods took place in the dry season, during which time chigger indices and infestation rates are typically lower as a consequence of the impact of dessication on the life cycle [1], although the proportion of positives would be expected to be similar [15]. The single positive pool was collected during the rainy season, when only 16 small mammals were trapped. Of these, 15 (94%) were infested with chiggers with much higher indices, and accounted for 718/1,446 (49%) of the total collected. The proportion of recognised human vector species, such as L. deliense, was very low at 10%. A previous study in Thailand identified high risk sites for human infection to be associated with a high proportion of known human vector species (Leptotrombidium spp.) [15]. The reason for this is uncertain. L. deliense is a host generalist, and present in a wide range of habitats with known seasonal fluctuations [15,32]. The factors that determine the density and diversity of different chigger species across habitats remain poorly known. The distribution of O. tsutsugamushi infection in chiggers is likely to be heterogeneous and change over time as has been described in the concept of "mite islands" [1].There is a possibility that certain habitats were under sampled due to a reliance on hunters. Rodent seropositivity at 35% was higher than that reported on average (at 30%) across all studies [1]. This suggests that despite the low rates of molecular evidence of O. tsutsugamushi in chiggers and small mammals, there is evidence of high rates of exposure to the pathogen. In small mammals the pathogen was detectable in blood for a mean of 97 days [33], until 8 weeks after the bite of an infected Leptotrombidium chigger [34], and in kidney tissue after 4 months [35]. In one study in Thailand IgG persisted for 10 months in animals removed from the wild and kept in captivity [36].
The lone positive chigger pool in this study consisted of mixed Walchia species, although the presence of L. deliense cannot be excluded as not all chiggers were identified. There is no evidence that Walchia acts as an O. tsutsugamushi vector for humans, but a small number of studies have identified O. tsutsugamushi in this genus [1,15] suggesting the potential to act as an intra-zootic vector.
There are a number of limitations to this study. Firstly collections took place only at 3 time points over an 18 month period and only once (October 2016) during the wet season.
In October 2016 only a small number of animals were trapped, predominantly due to unavailability of hunters due to the peak rice harvesting period. Not all rodent tissues were tested by PCR. Previous studies have demonstrated that O. tsutsugamushi positivity can vary between organ types, with spleen and lung tissue most frequently positive [15]. Seventeen percent (243/1446) of chiggers removed from small mammals were identified to species level. It is likely the overall diversity is under-represented.

Conclusion
Despite the low rates of O. tsutsutgamushi PCR positive vectors and hosts in this study, evidence from serological data confirms the presence of the pathogen in endemic areas.
Further studies are needed to identify the key vector species and ecological factors associated with high risk sites for human transmission in Laos. This will be especially important for planning the prevention and management of this neglected disease, given the evidence that the consequences of global warming, especially flooding, may increase disease burden [9].